Mature Anthers Research Articles

Anther wall and pollen development in Neotropical species-rich Miconia (Melastomataceae)

Miconia is one of the largest exclusively Neotropical genera, inserted in the tribe Miconieae. Although the monophyly of the tribe has recently been recognized, the delimitation of its genera is considered quite arbitrarily defined, mainly due to the great diversity combined with little morphological and genetic characterization of its members. Recent findings have associated this diversity with polyploidy, apomixis and hybridization, mechanisms commonly found in the group. The conventional delimitation of sections in Miconia, which has been largely based on stamen morphology, has proved to be undoubtedly artificial; nevertheless, the potential taxonomic significance of anther wall and pollen ontogenetic characters within the genera Miconia has been little explored. Hence, this study intended to fill that gap in our knowledge, by investigating the anther wall and pollen development in six closely related species of Miconia: M. albicans, M. fallax, M. latecrenata, M. paucidens, M. pepericarpa and M. stenostachya. Routine techniques for both light and electronic microscopy were used to examine anthers and pollen grains in several developmental stages. The species studied here share several character states, such as the remarkable monocotyledonous anther wall development, the persistent endothecium in the mature anthers with no cell wall thickenings, the bilocular dehiscent anther and the psilate pollen exine. This study also reports for the first time bisporangiate anthers, crystals and proteinoplasts in tapetal cells for Melastomataceae, and ultrastructural features of anther and pollen wall for Miconia.

Pectic arabinan side chains are essential for pollen cell wall integrity during pollen development

Pectin is a complex polysaccharide and an integral part of the primary plant cell wall and middle lamella, contributing to cell wall mechanical strength and cell adhesion. To understand the structure-function relationships of pectin in the cell wall, a set of transgenic potato lines with altered pectin composition was analysed. The expression of genes encoding enzymes involved in pectin acetylation, degradation of the rhamnogalacturonan backbone and type and length of neutral side chains, arabinan and galactan in particular, has been altered. Upon crossing of different transgenic lines, some transgenes were not transmitted to the next generation when these lines were used as a pollen donor, suggesting male sterility. Viability of mature pollen was severely decreased in potato lines with reduced pectic arabinan, but not in lines with altered galactan side chains. Anthers and pollen of different developmental stages were microscopically examined to study the phenotype in more detail. Scanning electron microscopy of flowers showed collapsed pollen grains in mature anthers and in earlier stages cytoplasmic protrusions at the site of the of kin pore, eventually leading to bursting of the pollen grain and leaking of the cytoplasm. This phenomenon is only observed after the microspores are released and the tapetum starts to degenerate. Timing of the phenotype indicates a role for pectic arabinan side chains during remodelling of the cell wall when the pollen grain is maturing and dehydrating.

Cloning and characterisation of a putative pollen‐specific polygalacturonase gene (CpPG1) differentially regulated during pollen development in zucchini (Cucurbita pepo L.)

Studies in zucchini (Cucurbita pepo L. spp. pepo) pollen have been limited to the viability and morphology of the mature pollen grain. The enzyme polygalacturonase (PG) is involved in pollen development and pollination in many species. In this work, we study anther and pollen development of C.pepo and present the cloning and characterisation of a putative PG CpPG1 (Accession no. HQ232488) from pollen cDNA in C.pepo. The predicted protein for CpPG1 has 416 amino acids, with a high homology to other pollen PGs, such as P22 from Oenothera organensis (76%) and PGA3 from Arabidopsis thaliana (73%). CpPG1 belongs to clade C, which comprises PGs expressed in pollen, and presents a 34 amino acid signal peptide for secretion towards the cell wall. DNA-blot analysis revealed that there are at least another two genes that code for PGs in C.pepo. The spatial and temporal accumulation of CpPG1 was studied by semi-quantitative- and qRT-PCR. In addition, mRNA was detected only in anthers, pollen and the rudimentary anthers of bisexual flowers (only present in some zucchini cultivars under certain environmental conditions that trigger anther development in the third whorl of female flowers). However, no expression was detected in cotyledons, stem or fruit. Furthermore, CpPG1 mRNA was accumulated throughout anther development, with the highest expression found in mature pollen. Similarly, exo-PG activity increased from immature anther stages to mature anthers and mature pollen. Overall, these data support the pollen specificity of this gene and suggest an involvement of CpPG1 in pollen development in C.pepo.

Diplospory and obligate apomixis in Miconia albicans (Miconieae, Melastomataceae) and an embryological comparison with its sexual congener M. chamissois

Apomixis, or asexual reproduction through seeds, has been reported for species of the tribe Miconieae, Melastomataceae, but details of the process have yet to be described. We analyzed and compared sporogenesis and gametogenesis in the apomictic Miconia albicans and the sexual M. chamissois. The results point to some differences between species, which were related to the apomictic process. In M. albicans microsporogenesis, problems during meiosis and degeneration of its products led to total pollen sterility, while M. chamissois presented normal bicellular pollen grains in the mature anther. The absence or abnormality of meiosis in M. albicans megasporogenesis led to the formation of an unreduced embryo sac and also to egg cell parthenogenesis, which gave rise to the apomictic embryo. Embryo and endosperm development were autonomous, resulting in seeds and fruits independent of pollination and fertilization. Thus, in this species, apomixis can be classified as diplosporic and obligate. In contrast, meiosis was as expected in the sexual M. chamissois, and led to the development of a reduced embryo sac. Despite the divergent pathways, many embryological characteristics were similar between the studied species and other Melastomataceae and they seem to be conservative character states for the family.

Cytological Investigation of Pollen Development in Sorghum Line with Male Sterility Induced by Sodium Ascorbate in Tissue Culture

Investigation of male sterility mutations is an effective approach for identification of genes involved in anther and pollen development. The comparison of “cytological phenotypes” of newly induced mutants with phenotypes determined by already known genes favors elucidation of genetic control of diverse microsporo- and gametogenesis stages. In this paper, we describe pollen development in the grain sorghum line Zh10-asc1 with mutation of male sterility. This line was obtained from callus culture treated by sodium ascorbate. A wide spectrum of abnormalities in microsporogenesis have been found, such as cytomixis, chromosomal laggards, chromosome disjunction, adhesion of chromosomes, disturbed cytokinesis, and others. In tapetum, the cells with one nucleus, with unequal nuclei, and with micronuclei have been observed. During pollen grain (PG) maturation abnormalities in starch accumulation and delay of development often took place. In mature anthers, a variety of pollen grain types have been revealed: fertile, of irregular shape, incompletely filled with starch, PGs delayed at the uni-nucleate or bi-nucleate gametophyte stages, with partially or fully degenerated contents, and with abnormal coloration. Variation in spectrum and the frequency of disturbances between the flowers of one and the same plant have been revealed. The reasons for significant genetic and epigenetic instability are discussed.

Cover Caption: Rice Anther Development

At the early stage of rice flower development, the lemma, palea, stamen and pistil primordia are first formed, as shown by the cover picture taken under an electron microscope. The stamen primordia further develop into mature anthers, with viable pollen inside. In this issue, Guo and Liu (pp. 967–978) summarize recent advances in molecular genetic studies of rice anther development and male fertility/sterility control. Research in this field has significant implications in rice genetic improvement.

Anther structure and pollen development in Melicoccus lepidopetalus (Sapindaceae): An evolutionary approach to dioecy in the family

Anther and pollen development in staminate and pistillate flowers of dioecious Melicoccus lepidopetalus (Sapindaceae) were examined by light and electron microscopy. Young anthers are similar in both types of flowers; they consist of epidermis, endothecium, two to four middle layers and a secretory tapetum. The microspore tetrads are tetrahedral. The mature anther in staminate flowers presents compressed epidermal cells and endothecium cells with fibrillar thickenings. A single locule is formed in the theca by dissolution of the septum and pollen grains are shed at two-celled stage. The mature anthers of pistillate flowers differ anatomically from those of staminate flowers. The epidermis is not compressed, the endothecium does not develop fibrillar thickenings, middle layers and tapetum are generally persisting, and the stomium is nonfunctional. Microspore degeneration begins after meiosis of microspore mother cells. At anthesis, uninucleate microspores and pollen grains with vegetative and generative nuclei with no cytokinesis are observed. Some pollen walls display an abnormal exine deposition, whereas others show a well formed exine, although both are devoid of intine. These results suggest that in the evolution towards unisexuality, the developmental differences of anther wall tissues and pollen grains between pistillate and staminate flowers might become more pronounced in a derived condition, such as dioecy.

Embryological studies of Oxalis debilis Kunth

The study of pollen and female gametophyte development of Oxalis debilis was carried out to elucidate the reasons for the absence of seed production in this species. The formation of an incomplete callosic wall separating the microspores of the tetrad was observed; therefore, cytoplasm connections were present between the microspores. Microspores of different sizes and with different grades of vacuolation were observed in the mature anther. Only few microspores divide mitotically and form the generative and vegetative cell. The cytoplasm of the vegetative cell may accumulate different substances in reserve. Pollen grains are 8–12 colpate, and the morphology and electron density of the exine varies. Because flowers in full anthesis have all ovules at the megasporocyte stage with the nucellar epidermis intact, fecundation could not occur. The production of only a few viable pollen grains and the absence of successful megagametophyte development prevent fertilization and account for the absence of fruits and seeds formation.

The pollen wall and tapetum are altered in the cytoplasmic male sterile line RC7 of Chinese cabbage (Brassica campestris ssp pekinensis)

Cytoplasmic male sterile line RC(7) of Chinese cabbage produces mature anthers without pollen. To understand the mechanisms involved, we examined the ultrastructural changes during development of the microspores. Development of microspores was not affected at the early tetrad stage. During the ring-vacuolated period, some large vacuoles appeared in the tapetum cells, making them larger, extending to the anther sac center during the monocyte period. At the same time, the tapetum degenerated as the microspores aborted, resulting in pollen-deficient anthers. As a result, the locules collapsed and the anthers shriveled. The callose was degraded in the pollen walls; abnormal deposits of electrodense material gave rise to irregular spike-shaped structures, rather than the characteristic rod-like shape of the B7 bacula. The internal intine wall of RC(7) was thinner than that of the B7 type. At the mitosis I microspore stage, the tapetum cells contained multiple plastids, with numerous small spherical plastoglobuli, and lipid bodies. Based on these observations, we suggest that RC(7) abortion may be due to mutated genes that normally regulate development of the pollen wall and cell walls in the RC(7) line.

Natural History of Maguire Primrose,Primula cusickianavar.Maguirei(Primulaceae)

Natural history information for rare plants can help land managers better understand the threats to extinction that a taxon may face. Our focus is on the natural history of Maguire primrose (Primula cusickiana var. maguirei, Primulaceae), an endemic, threatened plant found along a narrow corridor within a single canyon in northern Utah. We examined floral morphology, air temperature, relative humidity during flowering, dichogamy, blooming period, and visits from flying insects. As with most Primula, Maguire primrose displays distinct floral distyly. Within the distylous flowers, 17% of our samples had a timing difference in the maturation of anthers and stigmas, a trait not previously recorded in any other Primula species. Temperatures during the early-spring blooming period fluctuated widely between recorded minima below 0 °C and maxima above 33 °C. We captured 8 different species of flying insects visiting Maguire primrose flowers in air temperatures ranging from 6 to 15 °C. Bloom timing was not well synchronized between different canyon locations. Between different canyon locations, we observed only a small number of plants that overlapped in their flowering phenology. This threatened primrose variety has a cool, early-season blooming period, a dependence on visiting insects for outcrossing, and disjunct populations throughout the canyon, resulting in the potential for serious challenges to reproduction.

The fertility restorer gene, Rf2, for Lead Rice‐type cytoplasmic male sterility of rice encodes a mitochondrial glycine‐rich protein

Cytoplasmic male sterility (CMS) is associated with a mitochondrial mutation that causes an inability to produce fertile pollen. The fertility of CMS plants is restored in the presence of a nuclear-encoded fertility restorer (Rf) gene. In Lead Rice-type CMS, discovered in the indica variety 'Lead Rice', fertility of the CMS plant is restored by the single nuclear-encoded gene Rf2 in a gametophytic manner. We performed map-based cloning of Rf2, and proved that it encodes a protein consisting of 152 amino acids with a glycine-rich domain. Expression of Rf2 mRNA was detected in developing and mature anthers. An RF2-GFP fusion was shown to be targeted to mitochondria. Replacement of isoleucine by threonine at amino acid 78 of the RF2 protein was considered to be the cause of functional loss in the rf2 allele. As Rf2 does not encode a pentatricopeptide repeat protein, unlike a majority of previously identified Rf genes, the data from this study provide new insights into the mechanism for restoring fertility in CMS.

Rice pollen hybrid incompatibility caused by reciprocal gene loss of duplicated genes

Genetic incompatibility is a barrier contributing to species isolation and is caused by genetic interactions. We made a whole genome survey of two-way interacting loci acting within the gametophyte or zygote using independence tests of marker segregations in an F(2) population from an intersubspecific cross between O. sativa subspecies indica and japonica. We detected only one reproducible interaction, and identified paralogous hybrid incompatibility genes, DOPPELGANGER1 (DPL1) and DOPPELGANGER2 (DPL2), by positional cloning. Independent disruptions of DPL1 and DPL2 occurred in indica and japonica, respectively. DPLs encode highly conserved, plant-specific small proteins (∼10 kDa) and are highly expressed in mature anther. Pollen carrying two defective DPL alleles became nonfunctional and did not germinate, suggesting an essential role for DPLs in pollen germination. Although rice has many duplicated genes resulting from ancient whole genome duplication, the origin of this gene duplication was in recent small-scale gene duplication, occurring after Oryza-Brachypodium differentiation. Comparative analyses suggested the geographic and phylogenetic distribution of these two defective alleles, showing that loss-of-function mutations of DPL1 genes emerged multiple times in indica and its wild ancestor, O. rufipogon, and that the DPL2 gene defect is specific to japonica cultivars.

OsMYB103 is required for rice anther development by regulating tapetum development and exine formation

The Arabidopsis AtMYB103 gene is required for anther development, but whether the homologous gene in rice has the same role is unclear. Sequence analysis indicated that the rice OsMYB103 gene shares a high sequence similarity with AtMYB103. Therefore, we investigated the functional role of OsMYB103 in anther development using an RNAi approach. The OsMYB103 RNA transcript was expressed most abundantly in flowers, specifically in the tapetum, premeiotic pollen mother cells, and meiotic PMCs. OsMYB103-RNAi transgenic lines grew normally during their vegetative phase but displayed reduced male fertility, a phenotype that was associated with downregulated OsMYB103 transcript levels. Expression of OsMS2, an ortholog of the Arabidopsis AtMS2 gene, was also dramatically reduced in the transgenic plants. Knockdown of OsMYB103 led to defects in tapetum development, and most of the microspores in mature anthers lacked exines. Moreover, OsMYB103 could partially rescue the male sterility phenotype of an AtMYB103 knockout mutant ms188. Taken together, these results indicate that OsMYB103 does have an important role in rice tapetum and microspore development.

Two duplicate CYP704B1-homologous genes BnMs1 and BnMs2 are required for pollen exine formation and tapetal development in Brassica napus

S45A, a double recessive mutant at both the BnMs1 and BnMs2 loci in Brassica napus, produces no pollen in mature anthers and no seeds by self-fertilization. The BnMs1 and BnMs2 genes, which have redundant functions in the control of male fertility, are positioned on linkage groups N7 and N16, respectively, and are located at the same locus on Arabidopsis chromosome 1 based on collinearity between Arabidopsis and Brassica. Complementation tests indicated that one candidate gene, BnCYP704B1, a member of the cytochrome P450 family, can rescue male sterility. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of the developing anther showed that pollen-wall formation in the mutant was severely compromised, with a lack of sporopollenin or exine. The phenotype was first evident at the tetrad stage (stage 7) of anther development, coinciding with the maximum BnCYP704B1 mRNA accumulation observed in tapetal cells at stages 7-8 (haploid stage). TEM also suggested that development of the tapetum was seriously defective due to the disturbed lipid metabolism in the S45A mutant. A TUNEL assay indicated that the pattern of programmed cell death in the tapetum of the S45A mutant was defective. Lipid analysis showed that the total fatty acid content was reduced in the S45A mutant, indicating that BnCYP704B1 is involved in lipid metabolism. These data suggest that BnCYP704B1 participates in a vital tapetum-specific metabolic pathway that is not only involved in exine formation but is also required for basic tapetal cell development and function.

ATP-Binding Cassette Transporter G26 Is Required for Male Fertility and Pollen Exine Formation in Arabidopsis

The highly resistant biopolymer, sporopollenin, gives the outer wall (exine) of spores and pollen grains their unparalleled strength, shielding these structures from terrestrial stresses. Despite a limited understanding of the composition of sporopollenin, it appears that the synthesis of sporopollenin occurs in the tapetum and requires the transport of one or more sporopollenin constituents to the surface of developing microspores. Here, we describe ABCG26, a member of the ATP-binding cassette (ABC) transporter superfamily, which is required for pollen exine formation in Arabidopsis (Arabidopsis thaliana). abcg26 mutants are severely reduced in fertility, with most siliques failing to produce seeds by self-fertilization and mature anthers failing to release pollen. Transmission electron microscopy analyses revealed an absence of an exine wall on abcg26-1 mutant microspores. Phenotypic abnormalities in pollen wall formation were first apparent in early uninucleate microspores as a lack of exine formation and sporopollenin deposition. Additionally, the highest levels of ABCG26 mRNA were in the tapetum, during early pollen wall formation, sporopollenin biosynthesis, and sporopollenin deposition. Accumulations resembling the trilamellar lipidic coils in the abcg11 and abcg12 mutants defective in cuticular wax export were observed in the anther locules of abcg26 mutants. A yellow fluorescent protein-ABCG26 protein was localized to the endoplasmic reticulum and plasma membrane. Our results show that ABCG26 plays a critical role in exine formation and pollen development and are consistent with a model by which ABCG26 transports sporopollenin precursors across the tapetum plasma membrane into the locule for polymerization on developing microspore walls.

New Combinations in Habranthus (Amaryllidaceae) in Mexico and Southwestern U.S.A

New combinations in Habranthus Herb. (Amaryllidaceae) are made for four species that were previously treated as Zephyranthes Herb. (Z. arenicola Brandegee, Z. chichimeca T. M. Howard & S. Ogden, Z. conzattii Greenm., and Z. longifolia Hemsl.) and that are endemic to Mexico and the southwestern United States: H. arenicola (Brandegee) Flagg, G. Lom. Sm. & Meerow, H. chichimeca (T. M. Howard & S. Ogden) Flagg, G. Lom. Sm. & Meerow, H. conzattii (Greenm.) Flagg, G. Lom. Sm. & Meerow, and H. longifolius (Hemsl.) Flagg, G. Lom. Sm. & Meerow. The mature anthers, which are more or less horizontal and usually lunate or U-shaped, readily distinguish Habranthus from Zephyranthes, in which the mature anthers are vertical and relatively linear.

Microsporogenesis and microgametogenesis of Cardiospermum grandiflorum and Urvillea chacoensis (Sapindaceae, Paullinieae)

Microsporogenesis and microgametogenesis of two species, Cardiospermum grandiflorum Sw. and Urvillea chacoensis Hunz. (Sapindaceae, Paullinieae), were studied using light and transmission electron microscopy. Both species are monoecious, with staminate and hermaphrodite, although functionally pistillate, flowers. A comparative pollen-development study of these two floral morphs is reported. For the present study, five stages of pollen ontogeny were identified. The development of the anther wall is of basic type. Its wall consists of epidermis, endothecium, two middle layers and a uninucleate secretory tapetum. The microspore tetrads are tetrahedral. The mature anther in staminate flowers presents the endothecium with well developed fibrillar thickenings, remains of tapetal cells, a single locule formed in the theca by dissolution of the septum before anther dehiscence and two-celled pollen grains when shed. In functionally pistillate flowers, the mature anthers present remnants of the middle layers, tapetal cells without signs of degradation, the theca with two locules and pollen grains uni- or bicellular, some of them with the cytoplasm collapsed. These anthers are not dehiscent. It can be concluded that male sterility is characterised by failure to produce functional pollen grains, an event that would be associated with the persistence of tapetal cells. Ultrastructural analysis clearly shows the difference in tapetal cells between the two flower morphs.

Calreticulin expression and localization in plant cells during pollen–pistil interactions

In this report, the distributions of calreticulin (CRT) and its transcripts in Haemanthus pollen, pollen tubes, and somatic cells of the hollow pistil were studied. Immunoblot analysis of protein extracts from mature anthers, dry and germinated pollen, growing pollen tubes, and unpollinated/pollinated pistils revealed a strong expression of CRT. Both in vitro and in situ studies confirmed the presence of CRT mRNA and protein in pollen/pollen tubes and somatic cells of the pistil transmitting tract. The co-localization of these molecules in ER of these cells suggests that the rough ER is a site of CRT translation. In the pistil, accumulation of the protein in pollen tubes, transmitting tract epidermis (tte), and micropylar cells of the ovule (mc) was correlated with the increased level of exchangeable calcium. Therefore, CRT as a Ca(2+)-binding/buffering protein, may be involved in mechanism of regulation calcium homeostasis in these cells. The functional role of the protein in pollen-pistil interactions, apart from its postulated function in cellular Ca(2+) homeostasis, is discussed.

S locus-linked F-box genes expressed in anthers of Hordeum bulbosum

Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S (3) haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S (3)) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system.

AtTRP1 encodes a novel TPR protein that interacts with the ethylene receptor ERS1 and modulates development in Arabidopsis

Arabidopsis AtTRP1 is an orthologue of SlTPR1, a tomato tetratricopeptide repeat protein that interacts with the tomato ethylene receptors LeETR1 and NR in yeast 2-hybrid assays and in vitro, and modulates plant development. AtTRP1 is encoded by a single copy gene in the Arabidopsis genome, and is related to TCC1, a human protein that competes with Raf-1 for Ras binding, and distantly related to the immunophilin-like FK-binding proteins TWD1 and PAS1. The former is involved in auxin transport and the latter is translocated to the nucleus in response to auxin. AtTRP1 interacted preferentially with the Arabidopsis ethylene receptor ERS1 in yeast two-hybrid assays. This association was confirmed by in vivo co-immunoprecipitation. AtTRP1 promoter–GUS was highly expressed in vascular tissue, mature anthers, the abscission zone, and was induced by ACC. Overexpression of AtTRP1 in wild-type Arabidopsis resulted in dwarf plants with reduced fertility, altered leaf/silique morphology, and enhanced expression of the ethylene responsive gene AtChitB. Exogenous GA did not reverse the dwarf habit. Etiolated transgenic seedlings overexpressing AtTRP1 displayed enhanced sensitivity to low ACC and this was correlated with the transgene expression. Seedlings overexpressing AtTRP1 at high levels exhibited shortened and swollen hypocotyls, inhibited root growth, and an altered apical hook. Plants overexpressing AtTRP1 also showed a reduced response to exogenous IAA and altered expression of a subset of auxin early responsive genes. These results indicated that overexpression of AtTRP1 affects cross-talk between ethylene and auxin signalling and enhances some ethylene responses and alters some auxin responses. A model for AtTRP1 action is proposed.